Ca2+ signals regulate diverse functions in the developing nervous system, including proliferation and differentiation of neural stem cells (NSCs), migration of nascent neurons, and apoptosis. Although the role of Ca2+ signaling in the development of the nervous system is widely recognized, little is known about the mechanisms responsible for production of Ca2+ signals in NSCs. Among the various mechanisms by which cellular Ca2+ signals are generated, store-operated Ca2+ release-activated Ca2+ (CRAC) channels have emerged as a widespread pathway for regulating many Ca2+-dependent functions, including transcription, motility and proliferation. CRAC channels are encoded by the Orai genes, which give rise to three highly homologous proteins that are widely expressed in most tissues. Emerging evidence in our laboratory indicates that CRAC channels encoded by the canonical Orai1-STIM1 pathway are a major route of Ca2+ entry in NSCs. Moreover, Ca2+ influx through CRAC channels powerfully activates Ca2+-dependent gene expression mediated by the transcription factor, NFAT, and regulates the proliferation of NSCs. We hypothesize that CRAC channels are a key checkpoint for proliferation and differentiation of NSCs. The overall goal of the present proposal is to elucidate the molecular and functional properties of CRAC channels in NSCs and illuminate their role for gene expression, proliferation, and differentiation of NSCs. Using a combination of electrophysiological and biochemical approaches as well as genetically engineered mice lacking CRAC channel function, we will: 1) define the electrophysiolgical properties and molecular machinery of CRAC channels in NSCs, 2) investigate the gating mechanisms involved in the opening of the pore following binding of the ER Ca2+ sensor, STIM1, to CRAC channels, and, 3) illuminate the downstream functions of CRAC channels for gene expression, proliferation, and differentiation in NSCs. Findings from these studies will significantly deepen our understanding of Ca2+ signaling pathways in NSCs and the mechanisms regulating early neuronal development. In addition, by addressing the mechanisms of CRAC channel gating, our studies are likely to impact the design of new drugs against CRAC channels and provide a basis for new therapies for neurodegenerative diseases and brain injury.